Matching Items (140)
Description
In the 1970s James Watson recognized the inability of conventional DNA replication machinery to replicate the extreme termini of chromosomes known as telomeres. This inability is due to the requirement of a building block primer and was termed the end replication problem. Telomerase is nature's answer to the end replication problem. Telomerase is a ribonucleoprotein which extends telomeres through reverse transcriptase activity by reiteratively copying a short intrinsic RNA sequence to generate 3' telomeric extensions. Telomeres protect chromosomes from erosion of coding genes during replication, as well as differentiate native chromosome ends from double stranded breaks. However, controlled erosion of telomeres functions as a naturally occurring molecular clock limiting the replicative capacity of cells. Telomerase is over activated in many cancers, while inactivation leads to multiple lifespan limiting human diseases. In order to further study the interaction between telomerase RNA (TR) and telomerase reverse transcriptase protein (TERT), vertebrate TERT fragments were screened for solubility and purity following bacterial expression. Soluble fragments of medaka TERT including the RNA binding domain (TRBD) were identified. Recombinant medaka TRBD binds specifically to telomerase RNA CR4/CR5 region. Ribonucleotide and amino acid pairs in close proximity within the medaka telomerase RNA-protein complex were identified using photo-activated cross-linking in conjunction with mass spectrometry. The identified cross-linking amino acids were mapped on known crystal structures of TERTs to reveal the RNA interaction interface of TRBD. The identification of this RNA TERT interaction interface furthers the understanding of the telomerase complex at a molecular level and could be used for the targeted interruption of the telomerase complex as a potential cancer treatment.
ContributorsBley, Christopher James (Author) / Chen, Julian (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
Fluoroquinolone antibiotics have been known to cause severe, multisystem adverse side effects, termed fluoroquinolone toxicity (FQT). This toxicity syndrome can present with adverse effects that vary from individual to individual, including effects on the musculoskeletal and nervous systems, among others. The mechanism behind FQT in mammals is not known, although various possibilities have been investigated. Among the hypothesized FQT mechanisms, those that could potentially explain multisystem toxicity include off-target mammalian topoisomerase interactions, increased production of reactive oxygen species, oxidative stress, and oxidative damage, as well as metal chelating properties of FQs. This review presents relevant information on fluoroquinolone antibiotics and FQT and explores the mechanisms that have been proposed. A fluoroquinolone-induced increase in reactive oxygen species and subsequent oxidative stress and damage presents the strongest evidence to explain this multisystem toxicity syndrome. Understanding the mechanism of FQT in mammals is important to aid in the prevention and treatment of this condition.
ContributorsHall, Brooke Ashlyn (Author) / Redding, Kevin (Thesis director) / Wideman, Jeremy (Committee member) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
ABSTRACT The unique structural features of deoxyribonucleic acid (DNA) that are of considerable biological interest also make it a valuable engineering material. Perhaps the most useful property of DNA for molecular engineering is its ability to self-assemble into predictable, double helical secondary structures. These interactions are exploited to design a variety of DNA nanostructures, which can be organized into both discrete and periodic structures. This dissertation focuses on studying the dynamic behavior of DNA nanostructure recognition processes. The thermodynamics and kinetics of nanostructure binding are evaluated, with the intention of improving our ability to understand and control their assembly. Presented here are a series of studies toward this goal. First, multi-helical DNA nanostructures were used to investigate how the valency and arrangement of the connections between DNA nanostructures affect super-structure formation. The study revealed that both the number and the relative position of connections play a significant role in the stability of the final assembly. Next, several DNA nanostructures were designed to gain insight into how small changes to the nanostructure scaffolds, intended to vary their conformational flexibility, would affect their association equilibrium. This approach yielded quantitative information about the roles of enthalpy and entropy in the affinity of polyvalent DNA nanostructure interactions, which exhibit an intriguing compensating effect. Finally, a multi-helical DNA nanostructure was used as a model `chip' for the detection of a single stranded DNA target. The results revealed that the rate constant of hybridization is strongly dominated by a rate-limiting nucleation step.
ContributorsNangreave, Jeanette (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian J.-L. (Committee member) / Seo, Dong Kyun (Committee member) / Arizona State University (Publisher)
Created2011
Description
Rhodoferax antarcticus strain ANT.BR, a purple nonsulfur bacterium isolated from a microbial mat in Ross Island, Antarctica, is the first described anoxygenic phototrophic bacterium that is adapted to cold habitats and is the first beta-proteobacterium to undergo complete genome sequencing. R. antarcticus has unique absorption spectra and there are no obvious intracytoplasmic membranes in cells grown phototrophically, even under low light intensity. Analysis of the finished genome sequence reveals a single chromosome (3,809,266 bp) and a large plasmid (198,615 bp) that together harbor 4,262 putative genes. The genome contains two types of Rubiscos, Form IAq and Form II, which are known to exhibit quite different kinetic properties in other bacteria. The presence of multiple Rubisco forms could give R. antarcticus high metabolic flexibility in diverse environments. Annotation of the complete genome sequence along with previous experimental results predict the presence of structural genes for three types of light-harvesting (LH) complexes, LH I (B875), LH II (B800/850), and LH III (B800/820). There is evidence that expression of genes for the LH II complex might be inhibited when R. antarcticus is under low temperature and/or low light intensity. These interesting condition-dependent light-harvesting apparatuses and the control of their expression are very valuable for the further understanding of photosynthesis in cold environments. Finally, R. antarcticus exhibits a highly motile lifestyle. The genome content and organization of all putative polar flagella genes are characterized and discussed.
ContributorsZhao, Tingting, M.S (Author) / Touchman, Jeffrey (Thesis advisor) / Rosenberg, Michael (Committee member) / Redding, Kevin (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2011
Description
Cyanovirin-N (CV-N) is a naturally occurring lectin originally isolated from the cyanobacteria Nostoc ellipsosporum. This 11 kDa lectin is 101 amino acids long with two binding sites, one at each end of the protein. CV-N specifically binds to terminal Manα1-2Manα motifs on the branched, high mannose Man9 and Man8 glycosylations found on enveloped viruses including Ebola, Influenza, and HIV. wt-CVN has micromolar binding to soluble Manα1-2Manα and also inhibits HIV entry at low nanomolar concentrations. CV-N's high affinity and specificity for Manα1-2Manα makes it an excellent lectin to study for its glycan-specific properties. The long-term aim of this project is to make a variety of mutant CV-Ns to specifically bind other glycan targets. Such a set of lectins may be used as screening reagents to identify biomarkers and other glycan motifs of interest. As proof of concept, a T7 phage display library was constructed using P51G-m4-CVN genes mutated at positions 41, 44, 52, 53, 56, 74, and 76 in binding Domain B. Five CV-N mutants were selected from the library and expressed in BL21(DE3) E. coli. Two of the mutants, SSDGLQQ-P51Gm4-CVN and AAGRLSK-P51Gm4-CVN, were sufficiently stable for characterization and were examined by CD, Tm, ELISA, and glycan array. Both proteins have CD minima at approximately 213 nm, indicating largely β-sheet structure, and have Tm values greater than 40°C. ELISA against gp120 and RNase B demonstrate both proteins' ability to bind high mannose glycans. To more specifically determine the binding specificity of each protein, AAGRLSK-P51Gm4-CVN, SSDGLQQ-P51Gm4-CVN, wt-CVN, and P51G-m4-CVN were sent to the Consortium for Functional Glycomics (CFG) for glycan array analysis. AAGRLSK-P51Gm4-CVN, wt-CVN, and P51G-m4-CVN, have identical specificities for high mannose glycans containing terminal Manα1-2Manα. SSDGLQQ-P51Gm4-CVN binds to terminal GlcNAcα1-4Gal motifs and a subgroup of high mannose glycans bound by P51G-m4-CVN. SSDGLQQ-wt-CVN was produced to restore anti-HIV activity and has a high nanomolar EC50 value compared to wt-CVN's low nanomolar activity. Overall, these experiments show that CV-N Domain B can be mutated and retain specificity identical to wt-CVN or acquire new glycan specificities. This first generation information can be used to produce glycan-specific lectins for a variety of applications.
ContributorsRuben, Melissa (Author) / Ghirlanda, Giovanna (Thesis advisor) / Allen, James (Committee member) / Wachter, Rebekka (Committee member) / Arizona State University (Publisher)
Created2013
Description
The discovery of DNA helical structure opened the door of modern molecular biology. Ned Seeman utilized DNA as building block to construct different nanoscale materials, and introduced a new field, know as DNA nanotechnology. After several decades of development, different DNA structures had been created, with different dimension, different morphology and even with complex curvatures. In addition, after construction of enough amounts DNA structure candidates, DNA structure template, with excellent spatial addressability, had been used to direct the assembly of different nanomaterials, including nanoparticles and proteins, to produce different functional nanomaterials. However there are still many challenges to fabricate functional DNA nanostructures. The first difficulty is that the present finite sized template dimension is still very small, usually smaller than 100nm, which will limit the application for large amount of nanomaterials assembly or large sized nanomaterials assembly. Here we tried to solve this problem through developing a new method, superorigami, to construct finite sized DNA structure with much larger dimension, which can be as large as 500nm. The second problem will be explored the ability of DNA structure to assemble inorganic nanomaterials for novel photonic or electronic properties. Here we tried to utilize DNA Origami method to assemble AuNPs with controlled 3D spacial position for possible chiral photonic complex. We also tried to assemble SWNT with discrete length for possible field effect transistor device. In addition, we tried to mimic in vivo compartment with DNA structure to study internalized enzyme behavior. From our results, constructed DNA cage origami can protect encapsulated enzyme from degradation, and internalized enzyme activity can be boosted for up to 10 folds. In summary, DNA structure can serve as an ideal template for construction of functional nanomaterials with lots of possibilities to be explored.
ContributorsZhao, Zhao (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian (Committee member) / Seo, Dong-Kyun (Committee member) / Arizona State University (Publisher)
Created2013
Description
DNA has recently emerged as an extremely promising material to organize molecules on nanoscale. The reliability of base recognition, self-assembling behavior, and attractive structural properties of DNA are of unparalleled value in systems of this size. DNA scaffolds have already been used to organize a variety of molecules including nanoparticles and proteins. New protein-DNA bio-conjugation chemistries make it possible to precisely position proteins and other biomolecules on underlying DNA scaffolds, generating multi-biomolecule pathways with the ability to modulate inter-molecular interactions and the local environment. This dissertation focuses on studying the application of using DNA nanostructure to direct the self-assembly of other biomolecular networks to translate biochemical pathways to non-cellular environments. Presented here are a series of studies toward this application. First, a novel strategy utilized DNA origami as a scaffold to arrange spherical virus capsids into one-dimensional arrays with precise nanoscale positioning. This hierarchical self-assembly allows us to position the virus particles with unprecedented control and allows the future construction of integrated multi-component systems from biological scaffolds using the power of rationally engineered DNA nanostructures. Next, discrete glucose oxidase (GOx)/ horseradish peroxidase (HRP) enzyme pairs were organized on DNA origami tiles with controlled interenzyme spacing and position. This study revealed two different distance-dependent kinetic processes associated with the assembled enzyme pairs. Finally, a tweezer-like DNA nanodevice was designed and constructed to actuate the activity of an enzyme/cofactor pair. Using this approach, several cycles of externally controlled enzyme inhibition and activation were successfully demonstrated. This principle of responsive enzyme nanodevices may be used to regulate other types of enzymes and to introduce feedback or feed-forward control loops.
ContributorsLiu, Minghui (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Chen, Julian (Committee member) / Zhang, Peiming (Committee member) / Arizona State University (Publisher)
Created2013
Description
Since Darwin popularized the evolution theory in 1895, it has been completed and studied through the years. Starting in 1990s, evolution at molecular level has been used to discover functional molecules while studying the origin of functional molecules in nature by mimicing the natural selection process in laboratory. Along this line, my Ph.D. dissertation focuses on the in vitro selection of two important biomolecules, deoxynucleotide acid (DNA) and protein with binding properties. Chapter two focuses on in vitro selection of DNA. Aptamers are single-stranded nucleic acids that generated from a random pool and fold into stable three-dimensional structures with ligand binding sites that are complementary in shape and charge to a desired target. While aptamers have been selected to bind a wide range of targets, it is generally thought that these molecules are incapable of discriminating strongly alkaline proteins due to the attractive forces that govern oppositely charged polymers. By employing negative selection step to eliminate aptamers that bind with off-target through charge unselectively, an aptamer that binds with histone H4 protein with high specificity (>100 fold)was generated. Chapter four focuses on another functional molecule: protein. It is long believed that complex molecules with different function originated from simple progenitor proteins, but very little is known about this process. By employing a previously selected protein that binds and catalyzes ATP, which is the first and only protein that was evolved completely from random pool and has a unique α/β-fold protein scaffold, I fused random library to the C-terminus of this protein and evolved a multi-domain protein with decent properties. Also, in chapter 3, a unique bivalent molecule was generated by conjugating peptides that bind different sites on the protein with nucleic acids. By using the ligand interactions by nucleotide conjugates technique, off-the shelf peptide was transferred into high affinity protein capture reagents that mimic the recognition properties of natural antibodies. The designer synthetic antibody amplifies the binding affinity of the individual peptides by ∼1000-fold to bind Grb2 with a Kd of 2 nM, and functions with high selectivity in conventional pull-down assays from HeLa cell lysates.
ContributorsJiang, Bing (Author) / Chaput, John C (Thesis advisor) / Chen, Julian (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2013
Description
There is a critical need for the development of clean and efficient energy sources. Hydrogen is being explored as a viable alternative to fuels in current use, many of which have limited availability and detrimental byproducts. Biological photo-production of H2 could provide a potential energy source directly manufactured from water and sunlight. As a part of the photosynthetic electron transport chain (PETC) of the green algae Chlamydomonas reinhardtii, water is split via Photosystem II (PSII) and the electrons flow through a series of electron transfer cofactors in cytochrome b6f, plastocyanin and Photosystem I (PSI). The terminal electron acceptor of PSI is ferredoxin, from which electrons may be used to reduce NADP+ for metabolic purposes. Concomitant production of a H+ gradient allows production of energy for the cell. Under certain conditions and using the endogenous hydrogenase, excess protons and electrons from ferredoxin may be converted to molecular hydrogen. In this work it is demonstrated both that certain mutations near the quinone electron transfer cofactor in PSI can speed up electron transfer through the PETC, and also that a native [FeFe]-hydrogenase can be expressed in the C. reinhardtii chloroplast. Taken together, these research findings form the foundation for the design of a PSI-hydrogenase fusion for the direct and continuous photo-production of hydrogen in vivo.
ContributorsReifschneider, Kiera (Author) / Redding, Kevin (Thesis advisor) / Fromme, Petra (Committee member) / Jones, Anne (Committee member) / Arizona State University (Publisher)
Created2013
Description
DNA nanotechnology has been a rapidly growing research field in the recent decades, and there have been extensive efforts to construct various types of highly programmable and robust DNA nanostructures. Due to the advantage that DNA nanostructure can be used to organize biochemical molecules with precisely controlled spatial resolution, herein we used DNA nanostructure as a scaffold for biological applications. Targeted cell-cell interaction was reconstituted through a DNA scaffolded multivalent bispecific aptamer, which may lead to promising potentials in tumor therapeutics. In addition a synthetic vaccine was constructed using DNA nanostructure as a platform to assemble both model antigen and immunoadjuvant together, and strong antibody response was demonstrated in vivo, highlighting the potential of DNA nanostructures to serve as a new platform for vaccine construction, and therefore a DNA scaffolded hapten vaccine is further constructed and tested for its antibody response. Taken together, my research demonstrated the potential of DNA nanostructure to serve as a general platform for immunological applications.
ContributorsLiu, Xiaowei (Author) / Liu, Yan (Thesis advisor) / Chang, Yung (Thesis advisor) / Yan, Hao (Committee member) / Allen, James (Committee member) / Zhang, Peiming (Committee member) / Arizona State University (Publisher)
Created2012